恒温扩增微控流芯片技术快速检测关节置换术后感染病原菌的应用研究

发布时间：2018-06-15 16:49

本文选题：恒温扩增微控流芯片核酸分析技术 + 人工关节置换　；参考：《昆明医科大学》2017年硕士论文

【摘要】：人工关节置换(Artificial joint replacement,AJR)术后感染是人工关节置换术后最严重的并发症之一,是广大关节外科医生不可回避的问题,其诊断的关键是找到致病菌,一旦明确病原菌,便可确定诊断,就可以开始相应的治疗。目前临床上主要用于检测病原菌的方法是细菌分离培养,检测耗时长,众多因素容易造成假阴性结果的缺点易导致延误最佳治疗时机。恒温扩增微控流芯片核酸分析技术是分子生物学技术迅猛发展背景下应运而生的新型病原菌检测技术,具有检测快速、灵敏度高、准确性高、简便高效等优点。摈弃了传统聚合酶链反应(polymerase chain reaction,PCR)技术操作繁琐、温度要求严格、标本易污染的缺点。从标本处理开始到检出结果,全程只需要2小时。可以同时检测多种病原菌。恒温扩增微控流芯片核酸分析技术目前主要被应用于呼吸道病原菌的检测,在全国5所大型三甲医院进行的2518例标本检测中,准确率高达94.5%。由于此项技术是近两年的新兴技术,目前尚未应用于AJR术后感染病原菌的检测,国内外也无相关文献报道。本课题通过研究恒温扩增微控流芯片核酸分析技术在AJR术后感染病原菌检测中的效果,探讨此项技术的临床实用性及推广价值。毋庸置疑,随着科学技术的发展以及临床与科研的紧密结合,恒温扩增技术在病原菌的诊断中必定占有一席之地。[目的]通过分析恒温扩增微控流芯片核酸分析技术(Nucleic Acid Analysy Technique of Isothermal Amplification Microfluidic Chip,NAATIAMC)快速检测AJR术后感染病原菌的效果,探讨其临床实用性及推广价值。[方法]1.选取2016年1月至2017年2月昆明医科大学第一附属医院骨科及呈贡分院骨科收治的疑似人工关节置换术后感染的28例患者为研究对象,其中男性12例,女性16例,年龄64.5±13.1岁(37～83岁);人工膝关节置换术后8例,人工髋关节置换术后20例。2.将采集到的破溃创口分泌物或关节穿刺液、术中假体周围组织碾磨液标本分为两组,各取5ml。同时采用恒温扩增微控流芯片核酸分析技术(实验组)和细菌分离培养法(对照组)进行病原菌检测/培养。3.记录实验组和对照组检测/培养出病原菌的种类及耗时,列出病原菌检测/培养结果四格表。通过一致性的假设检验比较两组数据结果的一致性;采用χ2检验,比较两组阳性率,p0.05,表示差异有统计学意义。[结果]1.实验组检测结果:24例阳性,4例阴性;对照组培养结果:17例阳性,11例阴性。实验组检测结果为阳性的24例标本中有7例行细菌分离培养结果为阴性;实验组中检测结果为阴性的4例标本,行细菌分离培养同样为阴性。2.实验组耗时约2h,对照组耗时约4-7d。3.实验组检测出的病原菌以金黄色葡萄球菌最多见(11株,39.3%),其次是大肠埃希菌(5株,18%)、耐甲氧西林葡萄球菌(3株,10.7%)、结核杆菌(3株,10.7%),肺炎克雷伯菌(2株,7.1%)、铜绿假单胞菌(2株,7.1%),鲍曼不动杆菌(2株,7.1%)。对照组培养出的病原菌以金黄色葡萄球菌最多见(8株,40%),其次是大肠埃希菌(5株,25%)、耐甲氧西林葡萄球菌(3株,15%)、肺炎克雷伯菌(2株,10%)、铜绿假单胞菌(1株,5%),鲍曼不动杆菌(1株,5%)。4.实验组恒温扩增微控流芯片核酸分析技术检测阳性率为85.7%(24/28),高于对照组细菌培养阳性率60.7%(17/28),差异有统计学意义(χ2=4.462,p0.05)。Kappa 值为 0.66,一致性较好。[结论]1.恒温扩增微控流芯片核酸分析技术检测人工关节置换术后感染病原菌与细菌分离培养相比有较高的阳性检出率,可有效的避免传统细菌分离培养结果的假阴性。并且在耗时和效率方面较传统的细菌分离培养相比具有明显的优势。2.资料中,人工关节置换术后假体周围感染病原菌以金黄色葡萄球菌等革兰氏阳性菌为主,其次是革兰氏阴性菌。3.本课题研究中,恒温扩增微控流芯片核酸分析技术对于结核杆菌的检测较细菌分离培养相比有一定的优势,显示了良好的应用前景。综上所述,恒温扩增微控流芯片核酸分析技术可适用于人工关节置换术后假体周围感染病原菌的检测,并具有便捷、快速及高阳性检出率,可早期诊断病原菌、指导有效治疗,从而缩短了抗感染治疗的时间,增进了患者的快速康复,因此,具有较高的临床推广价值。
[Abstract]:Infection after artificial joint replacement (Artificial joint replacement, AJR) is one of the most serious complications after artificial joint replacement. It is an unavoidable problem for Joint Surgeons. The key to diagnosis is to find pathogenic bacteria. Once the pathogenic bacteria are identified, the diagnosis can be determined and the corresponding treatment can be started. The methods used to detect pathogenic bacteria are bacteria isolation and culture, long time consuming, and many factors that cause false negative results easily lead to the best time to delay the best treatment. Speed, high sensitivity, high accuracy, simple and efficient and so on. It discarded the shortcomings of the traditional polymerase chain reaction (polymerase chain reaction, PCR) technique with tedious operation, strict temperature requirement, and easy to pollute the specimen. From the beginning of the specimen treatment to the detection result, the whole course only needs 2 small. At present, the technique of chip nucleic acid analysis is mainly used for the detection of respiratory pathogenic bacteria. In 2518 samples of 5 large three A hospitals in China, the accuracy is up to 94.5%. because this technique is a new technology in the past two years. It has not been applied to the detection of pathogenic bacteria after AJR, and there is no related literature at home and abroad. The effect of nucleic acid analysis on microcontrolled flow microchip nucleic acid analysis in the detection of pathogenic bacteria after AJR was studied. The clinical practicality and popularization value of this technique were discussed. With the development of science and technology and the close combination of clinical and scientific research, the technology of constant temperature amplification must have one seat in the diagnosis of pathogenic bacteria. [Objective] [Objective] to explore the clinical practicality and popularization value by analyzing the effect of Nucleic Acid Analysy Technique of Isothermal Amplification Microfluidic Chip, NAATIAMC for rapid detection of pathogenic bacteria after AJR operation, and to explore the value of its clinical practicability and popularization. [square method]1.] selected Kunming Medical University from January 2016 to February 2017. In the Department of orthopedics and Chenggong branch of the First Affiliated Hospital of the First Affiliated Hospital, 28 patients with suspected artificial joint replacement were studied, including 12 male, 16 female, 64.5 + 13.1 years old (37~83 years old), 8 cases after artificial knee replacement and 20 cases of.2. after artificial hip replacement. The puncture fluid was divided into two groups. The specimens were divided into two groups, each of which was taken by the constant temperature amplification microcontrol flow chip nucleic acid analysis technique (experimental group) and the bacterial isolation and culture method (control group). The pathogen detection / culture.3. records were recorded in the experimental group and the control group. The species and time consuming of the pathogens were detected / cultured, and the pathogen detection was listed. The results of the results were compared between the two groups and the consistency of the results of the four groups was tested by the hypothesis of consistency. The positive rates of the two groups were compared with the x 2 test, P0.05, indicating that the difference was statistically significant. [results the test results of the]1. experimental group, 24 cases positive, 4 negative, 17 cases positive and 11 negative. The test group was positive 2. The results of bacterial isolation and culture were negative in 7 of the 4 specimens. In the experimental group, 4 specimens with negative test results, bacterial isolation and culture were same as 2h in the negative.2. experimental group, and the control group took the time of 4-7d.3. test to detect Staphylococcus aureus most (11, 39.3%), followed by Escherichia coli (5, 18%). Methicillin resistant Staphylococcus (3 strains, 10.7%), Mycobacterium tuberculosis (3 strains, 10.7%), Klebsiella pneumoniae (2 strains, 7.1%), Pseudomonas aeruginosa (2 strains, 7.1%), Acinetobacter Bauman (2, 7.1%). The most common bacteria in the control group were Staphylococcus aureus (8, 40%), followed by Escherichia coli (5, 25%), methicillin resistant Staphylococcus. 15%) Klebsiella pneumoniae (2 strains, 10%), Pseudomonas aeruginosa (1 strains, 5%), Acinetobacter Bauman (1, 5%).4. experimental group with constant temperature amplification microcontrolled flow chip nucleic acid analysis technique, the positive rate was 85.7% (24/28), higher than the control group of bacterial culture positive rate 60.7% (17 /28), the difference was statistically significant (chi 2=4.462, P0.05).Kappa value was 0.66, conformance comparison [conclusion]1. isothermal amplification microcontrolled flow chip nucleic acid analysis technology has a higher positive rate compared with bacteria isolation and culture after artificial joint replacement, which can effectively avoid false negative of traditional bacterial isolation and culture results, and is more obvious in time and efficiency than traditional bacterial isolation and culture. In the predominant.2. data, the pathogenic bacteria around the prosthesis were mainly Gram-positive bacteria such as Staphylococcus aureus, followed by the Gram-negative bacteria.3., and the nucleic acid analysis technology of the isothermal amplification micro control flow chip has a certain advantage compared with the bacterial isolation and culture. In summary, the nucleic acid analysis technology of isothermal amplification micro control flow chip can be applied to the detection of pathogenic bacteria around the prosthesis after artificial joint replacement, and it has the convenience, rapid and high positive detection rate. It can diagnose the pathogenic bacteria early and guide effective treatment, thus shortening the time of anti infection treatment and promoting the patient. Therefore, it has high clinical value.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687.4

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